The progressive salinization of agricultural soils poses a major limitation for the growth and productivity of crop plants. Although engineering technologies involving drainage and supply of high quality water have been developed to overcome this problem, the existing methods are extremely costly and time-consuming. In many instances, due to the increased need for extensive agriculture, neither improved irrigation efficiency nor the installation of drainage systems is applicable. Moreover, in the arid and semi-arid regions of the world water evaporation exceeds precipitation. These soils are inherently high in salt and require vast amounts of irrigation to become productive. Since irrigation water contains dissolved salts and minerals, application of water further compounds the salinity problem.
Current attempts to enhance the salinity tolerance of model and crop plants are based on conventional breeding and selection of resistant variants. However, such breeding techniques typically require years to develop, are labor intensive and expensive. Moreover, thus far, these breeding and selecting strategies did not result in the mass production of tolerant varieties, suggesting that conventional breeding practices are not sufficient.
An alternative and attractive approach involves the genetic engineering of transgenic crops having enhanced salt tolerance. In recent years, advances in molecular biology have allowed mankind to manipulate the genetic complement of animals and plants. Genetic engineering of plants entails the isolation and manipulation of genetic material (typically in the form of DNA or RNA) and the subsequent introduction of the genetic material into plants. Such technology has led to the development of plants with increased pest resistance, plants that are capable of expressing pharmaceuticals and other chemicals and plants that express beneficial agricultural traits.
The primary negative effects imposed on plants by saline soil are the generation of osmotic imbalance due to ion uptake by the plant cell, and the toxicity of the ions. Sodium ions are toxic to plants due to their adverse effect on potassium nutrition, cytosolic enzyme activities, photosynthesis and metabolism. Different mechanisms function cooperatively to prevent accumulation of sodium ions (Na+) in the cytoplasm of plant cells, namely restriction of Na+ influx, active Na+ efflux and compartmentalization of Na+ in the vacuole. A comparison of ion distribution in cells and tissues of various plant species indicates that a primary characteristic of salt-tolerant plants is their ability to exclude sodium out of the cell or to take up sodium and sequester it in the cell vacuoles (Niu, X., et al., 1995 Plant Physiol. 109, 735-742). Although there is a wide spectrum of plant responses to salinity that are defined by a range of adaptations at the cellular and the whole plant levels, the mechanism of sodium transport appears to be fundamentally similar. At the cellular level, sodium ions are extruded by plasma membrane Na+/H+ antiporters that are energized by the proton gradient generated by the plasma membrane H+-ATPases (PM H+-ATPases). Cytoplasmic Na+ may also be compartmentalized by vacuolar Na+/H+ antiporters. These transporters are energized by the proton gradient generated by the vacuolar H+-ATPase and H+-PPiase.
A mechanism that may underlie the adaptation or tolerance of plants to osmotic stresses is the accumulation of compatible, low molecular weight osmolytes such as sugar alcohols, special amino acids, and glycinebetaine. Recently, a transgenic study has demonstrated that accumulation of the sugar alcohol mannitol in transgenic tobacco conferred protection against salt stress (Tarczynski M C, et al., (1993). Science 259: 508-510). Two recent studies using a transgenic approach have demonstrated that metabolic engineering of the glycinebetaine biosynthesis pathway is not only possible but also may eventually lead to production of stress-tolerant plants (Holmstrom K O, et al., (1994) Plant J 6: 749-758).
In addition to accumulation of low molecular weight compounds, a large set of genes is subjected to transcriptional regulation, which leads to the accumulation of new proteins in vegetative tissue of plants under osmotic stress conditions. The expression levels of a number of genes have been reported to be correlated with desiccation, salt, or cold tolerance of different plant varieties of the same species. It is generally assumed that stress-induced proteins might play a role in tolerance, but the functions of many stress-responsive genes are unknown. Detecting stress-responsive genes as well as elucidating their function will not only advance our understanding of plant adaptation and tolerance to environmental stresses, but also may provide important information for designing new strategies and tools for crop improvement (Chandler P M and Robertson M., (1994) Annu Rev Plant Physiol Plant Mol Biol 45: 113-141).
The response of plants to salt stress has previously been studied in model plant species with sequenced genomes, including Arabidopsis thaliana (Consortium S. (2000) Nature 408: 796-815) and rice (Goff S A, et al. (2002) Science 296: 92-100; Yu J, et al. (2002) Science 296: 79-92). Differential genomic screens carried out in Arabidopsis and rice have shown that plants respond to salt stress by up-regulation of a large number of genes involved in diverse physiological functions.
For example, a homologue of sodium antiporter (AtNhx1) from the salt-sensitive plant Arabidopsis thaliana has been identified and characterized. Over expression of AtNhx1 in Arabidopsis as well as in fusion yeast shows increased salt tolerance due to better performance of salt compartmentation into the vacuole (Apse M P, et al. (1999) Science 285: 1256-1258). Zhang et al have shown that over expression of vacuolar Na+/H+ antiporter in A. thaliana and tomato plants led to a significant enhancement in salinity tolerance (Zhang H X & Blumwald E (2001) Nature Biotechnology 19: 765-768). Shi et al demonstrated that over expression of Na+/H+ antiporter SOS1 in plant plasma membranes improves salinity tolerance in A. thaliana, suggesting that a plasma membrane-type Na+/H+ antiporter is essential for plant salt tolerance. (Shi H, Lee B H & Zhu J K (2003) Nat Biotechnology 21: 81-85).
US Patent Application No. 20040040054 discloses polynucleotides encoding plant Na+/H+ antiporter polypeptides isolated from Physcomitrella patens and methods of applying these plant polypeptides to the identification, prevention, and/or conferment of resistance to various plant diseases and/or disorders, particularly environmental stress tolerance in plants, specifically salt stress.
US Patent Application No. 2002178464 discloses transgenic plants transformed with exogenous nucleic acid which alters expression of vacuolar pyrophosphatases in the plant, wherein the transgenic plants are tolerant to a salt. Specifically, the exogenous nucleotide encodes a vacuolar pyrophosphatase H+ pump, AVP1.
International Patent Application No. WO 03/031631 discloses nucleic acids and nucleic acid fragments encoding amino acid sequences for salt stress-inducible proteins, protein phosphatases mediating salt adaptation in plants, plasma membrane sodium/proton antiporters, salt-associated proteins, glutathione peroxidase homologs associated with response to saline stress in plants, and early salt-responding enzymes such as glucose 6-phosphate 1 dehydrogenase and fructose-biphosphate aldolase in plants and the use thereof for, inter alia, modification of plant tolerance to environmental stresses and osmotic stresses such as salt stress, modification of plant capacity to survive salt shocks, modification of compartmentalization of sodium in plants, for example into the plant cell vacuole, modification of sodium ion influx and/or efflux, modification of plant recovery after exposure to salt stress, and modification of plant metabolism under salt stress.
U.S. Pat. No. 5,981,842 discloses a method of producing a cereal plant cell or protoplast useful for regeneration of a water stress or salt stress tolerant cereal plant by transforming the cereal plant cell or protoplast with a nucleic acid encoding a late embryogenesis abundant (LEA) protein. An LEA protein gene, HVA1, from barley (Hordeum vulgare L.) was transformed into rice (Oryza sativa L.) plants. The resulting transgenic rice plants accumulate the HVA1 protein in both leaves and roots. Transgenic rice plants showed significantly increased tolerance to water stress (drought) and salt stress. These studies demonstrate that, using a combination of breeding strategies and genetic manipulation, it is possible to generate plant crops having enhanced salt tolerance. However, all of the aforementioned methods rely on the isolation, characterization and over expression of genes from salt sensitive plant sources, and accordingly the success of such approaches relies on the expression of the plant genetic material, and the stability of the encoded proteins, in a salt environment.
Exceptionally salt tolerant (halotolerant) organisms may provide useful for identification of basic mechanisms that enhance salinity tolerance. A special example of adaptation to variable saline conditions is the unicellular green algae Dunaliella, a dominant organism in many saline environments, which can adapt to practically the entire range of salinities. Dunaliella responds to salt stress by massive accumulation of glycerol (its internal osmotic element), enhanced elimination of Na+ ions, and accumulation of distinct proteins (Pick U et al. In A Lauchli, U Luthge, Eds, Salinity: Environment-Plants Molecules, Ed Acad. Pub. Dordrecht. Kluwer, pp 97-112, 2002). Since the cells of this genus do not possess a rigid cell wall, they respond to changes in salt concentration by rapid alterations in cell volume and then return to their original volume as a result of adjustments in the amounts of intracellular ions and glycerol. It has been reported that the adaptation to extreme salinity involves short-term and long-term responses. The former include osmotic adjustment by accumulation of large amounts of intracellular glycerol and efficient elimination of Na+ ions by plasma membrane transporters. The latter involves synthesis of two extrinsic plasma membrane proteins, a carbonic anhydrase and a transferrin-like protein. These proteins are associated with acquisition of CO2 and Fe, respectively, whose availability is diminished by high salinity. In addition, Ajalov et al reported on the isolation of a 64 kDA and 28 kDA salt-induced polypeptides from Dunaliella salina (Ajalov et al. (1996), Biochemical Society Transactions, 24(4), 5345).
Due to its remarkable ability to adapt to highly saline conditions, Dunaliella serves as a valuable model for the identification of basic mechanisms of salinity tolerance, and as a source for useful salt responsive genes.
The success of current plant breeding strategies which are based on genetic manipulation of genes from plant sources has been constrained due to the limited capability of many plants, specifically crop plants to adapt to saline conditions. There remains a need in the art to develop genetic engineering approaches that are superior to current techniques, and that would yield transgenic plants having high salt tolerance that are capable of growing in conditions of high salinity.